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J Struct Biol. 2015 Dec;192(3):510-518. doi: 10.1016/j.jsb.2015.10.014. Epub 2015 Oct 19.

Structural insights on mouse L-threonine dehydrogenase: A regulatory role of Arg180 in catalysis.

Author information

1
Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis and School of Life Sciences, Anhui University, Hefei, Anhui 230601, China. Electronic address: chaohe@ahu.edu.cn.
2
Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis and School of Life Sciences, Anhui University, Hefei, Anhui 230601, China.
3
Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, China.
4
Institute of Cancer Stem Cell, Dalian Medical University, Dalian, Liaoning 116044, China.

Abstract

Mouse L-threonine dehydrogenase (mTDH), which belongs to the short-chain dehydrogenase/reductase (SDR) superfamily and mediates threonine catabolism, plays pivotal roles in both powerful biosynthesis and signaling in mouse stem cells and has a regulatory residue Arg180. Here we determined three crystal structures of mTDH: wild-type (WT) in the apo form; in complex with NAD(+) and a substrate analog, glycerol, or with only NAD(+); as well as the R180K variant with NAD(+). This is the first description of a structure for mammalian SDR-type TDH. Structural comparison revealed the structural basis for SDR-type TDH catalysis remains strictly conserved in bacteria and mammals. Kinetic enzyme assays, and isothermal titration calorimetry (ITC) measurements indicated the R180K mutation has little effect on NAD(+) binding affinity, whereas affects the substrate's affinity for the enzyme. The crystal structure of R180K with NAD(+), biochemical and spectroscopic studies suggested that the R180K mutant should bind NAD(+) in a similar way and have a similar folding to the WT. However, the R180K variant may have difficulty adopting the closed form due to reduced interaction of residue 180 with a loop which connects a key position for mTDH switching between the closed and open forms in mTDH catalysis, and thereby exhibited a significantly decreased kcat/Km value toward the substrate, L-Thr. In sum, our results suggest that activity of GalE-like TDH can be regulated by remote interaction, such as hydrogen bonding and hydrophobic interaction around the Arg180 of mTDH.

KEYWORDS:

Arg180; Crystal structure; Dehydrogenase; Enzyme kinetics; Enzyme mutation; Mouse; Nicotinamide adenine dinucleotide (NAD); l-Threonine

PMID:
26492815
DOI:
10.1016/j.jsb.2015.10.014
[Indexed for MEDLINE]

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